Infections by various yeast species have become recognized as a cause of disease and ultimate death in many types of patients, including cancer patients and immunocompromised patients. Candidemia has increased over the past decade with Candida albicans being the most often isolated organism. Other species frequently isolated from infected patients include Candida glabrata, Candida tropicalis, and Candida krusei. Resistance to therapeutics, including the polyenes and the azoles, is common in patients infected with various yeast species.
A sensitive method using PCR assays which are based on the detection of yeast DNA in human body fluids or tissue samples is herein described. The use of realtime PCR (RT-PCR) for detection of yeast infections increases the reliability of PCR results.
In one embodiment, a method of identifying a specific yeast species in patient tissue or body fluid is provided. The method comprises the steps of extracting and recovering DNA of the yeast species from the patient tissue or body fluid, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the yeast species, and specifically identifying the yeast species.
In this embodiment, 1) the amplifying step can be performed with primers that hybridize to the DNA, 2) the body fluids can be selected from the group consisting of urine, nasal secretions, nasal washes, bronchial lavages, bronchial washes, spinal fluid, sputum, gastric secretions, seminal fluid, other reproductive tract secretions, lymph fluid, whole blood, serum, and plasma, 3) the DNA can be amplified using PCR, 4) the PCR can be real-time PCR, 5) the probe can be fluorescently labeled, 6) the yeast species can be selected from the group consisting of Candida albicans, Candida glabrata, Candida kruseii, and Candida tropicalis, 7) the probe, a forward primer, and a reverse primer can be used during the amplification step and the probe comprises the sequence of SEQ ID NO: 1, the forward primer comprises the sequence of SEQ ID NO: 2, and the reverse primer comprises the sequence of SEQ ID NO: 3, 8) the probe, a forward primer, and a reverse primer can be used during the amplification step and the probe comprises the sequence of SEQ ID NO: 4, the forward primer comprises the sequence of SEQ ID NO: 5, and the reverse primer comprises the sequence of SEQ ID NO: 6, 9) the probe, a forward primer, and a reverse primer can be used during the amplification step and the probe comprises the sequence of SEQ ID NO: 7, the forward primer comprises the sequence of SEQ ID NO: 8, and the reverse primer comprises the sequence of SEQ ID NO: 9, 10) the probe, a forward primer, and a reverse primer can be used during the amplification step and the probe comprises the sequence of SEQ ID NO: 10, the forward primer comprises the sequence of SEQ ID NO: 11, and the reverse primer comprises the sequence of SEQ ID NO: 12, 11) the amplified sequence can be internal transcribed spacer regions of nuclear ribosomal DNA, and/or 12) the probe can be bound to a bead dyed with a fluorochrome. Any applicable combination of 1 through 12 is also contemplated.
In another embodiment, a method of identifying a yeast mycotoxin in patient tissue or body fluid is provided. The method comprises the steps of extracting and recovering the mycotoxin from the patient tissue or body fluid, contacting the mycotoxin with an antibody directed against the mycotoxin, and identifying the myocotoxin. In another embodiment, the method can further comprise the step of quantifying the mycotoxin. In either of these embodiments, 1) the body fluid can be selected from the group consisting of urine, nasal secretions, nasal washes, bronchial lavages, bronchial washes, spinal fluid, sputum, gastric secretions, seminal fluid, other reproductive tract secretions, lymph fluid, whole blood, scrum, and plasma, 2) the mycotoxin can be selected from the group consisting of a gliotoxin and a patulin, 3) the tissue can be derived from a patient tissue biopsy and can be in a 10% formalin solution or can be in a paraffin block, and/or 4) the antibody can be bound to a bead dyed with a fluorochrome. Any applicable combination of 1 through 4 is also contemplated.
In yet another embodiment, a method of determining if a patient is at risk for or has developed a disease state related to a yeast infection is provided. The method comprises the steps of extracting and recovering a yeast mycotoxin from a tissue or body fluid of the patient, contacting the mycotoxin with an antibody directed against the toxin, identifying the mycotoxin, and determining if the patient is at risk for or has developed the disease state related to the yeast infection. In another embodiment, the method can further comprise the step of developing an effective treatment regimen for the patient.
In either of the methods in the immediately preceding paragraph, 1) the body fluid can be selected from the group consisting of urine, nasal secretions, nasal washes, bronchial lavages, bronchial washes, spinal fluid, sputum, gastric secretions, seminal fluid, other reproductive tract secretions, lymph fluid, whole blood, serum, and plasma, 2) the mycotoxin can be selected from the group consisting of a gliotoxin and a patulin, 3) the tissue can be derived from a patient tissue biopsy and can be in a 10% formalin solution or can be in a paraffin block, and/or 4) the antibody can be bound to a bead dyed with a fluorochrome. Any applicable combination of 1 through 4 is also contemplated.
In still another embodiment, a method of determining if a patient is at risk for or has developed a disease state related to a yeast infection is provided. The method comprises the steps of extracting and recovering DNA of a specific yeast species from a tissue or body fluid of the patient, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the yeast species, and specifically identifying the yeast species. In another embodiment, the method can further comprise the step of developing an effective treatment regimen for the patient. In these two embodiments, the probe can be bound to a bead dyed with a fluorochrome.
In another illustrative embodiment, a kit is provided comprising a purified nucleic acid with a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 12 or with a complement of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 12.
In yet another illustrative embodiment, a kit is provided comprising components for the extraction and recovery of a yeast mycotoxin from body fluid or tissue of a patient. The kit can further comprise components for identification of the mycotoxin. The components for identification of the mycotoxin can include beads dyed with a fluorochrome and coupled to antibodies to the mycotoxin or to the mycotoxin or to a mycotoxin antigen.
In another embodiment, a purified nucleic acid is provided comprising a sequence of SEQ ID NO: 1 to SEQ ID NO: 12 or a sequence that hybridizes under highly stringent conditions to a sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 12.
In still another embodiment, a purified nucleic acid is provided comprising a complement of a sequence of SEQ ID NO: 1 to SEQ ID NO: 12 or a sequence that hybridizes under highly stringent conditions to a complement of a sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 12.
In another aspect, a method of detecting an antibody to a mycotoxin in a patient body fluid is provided. The method comprises the steps of contacting the patient body fluid with a mycotoxin or a mycotoxin antigen coupled to a bead wherein the bead is dyed with a fluorochrome, and detecting the antibody.
In yet another aspect, a method of identifying a yeast species in a patient tissue or body fluid is provided. The method comprises the steps of identifying a yeast mycotoxin in a patient tissue or body fluid, and specifically identifying a yeast species in the mycotoxin positive patient tissue or body fluid. The method can further comprise the steps of extracting and recovering the mycotoxin from the patient tissue or body fluid, and contacting the mycotoxin with an antibody directed against the mycotoxin. The method can further comprise the steps of extracting and recovering DNA of the yeast species from the patient tissue or body fluid, amplifying the DNA, and hybridizing a probe to the DNA to specifically identify the yeast species.
In any of the embodiments in the immediately preceding paragraph, 1) the amplifying step can be performed with primers that hybridize to the DNA, 2) the body fluids can be selected from the group consisting of urine, nasal secretions, nasal washes, bronchial lavages, bronchial washes, spinal fluid, sputum, gastric secretions, seminal fluid, other reproductive tract secretions, lymph fluid, whole blood, serum, and plasma, 3) the DNA can be amplified using PCR, 4) the PCR can be real-time PCR, 5) the probe can be fluorescently labeled, 6) the yeast species can be selected from the group consisting of Candida albicans, Candida glabrata, Candida kruseii, and Candida tropicalis, 7) the amplified sequence can be internal transcribed spacer regions of nuclear ribosomal DNA, 8) the probe can be bound to a bead dyed with a fluorochrome, 9) the method can further comprise the step of quantifying the mycotoxin, 10) the mycotoxin can be selected from the group consisting of a gliotoxin and a patulin, 11) the tissue can be derived from a patient tissue biopsy and can be in a 10% formalin solution or is in a paraffin block, and/or 12) the antibody can be bound to a bead dyed with a fluorochrome. Any applicable combination of 1 through 12 is also contemplated.